Abstract
Flunixin meglumine is a nonsteroidal anti-inflammatory drug approved to manage pyrexia associated with swine respiratory disease. In the United States, no analgesic drugs are approved for use in swine by the FDA, although they are needed to manage painful conditions. This study evaluated the pharmacokinetics and relative bioavailability of intranasal versus intramuscular flunixin in grower pigs. Six pigs received 2.2 mg/kg flunixin either intranasally via atomizer or intramuscularly before receiving flunixin via the opposite route following a 5-day washout period. Plasma samples were collected over 60 h and analysed using ultra-performance liquid chromatography and tandem mass spectrometry to detect flunixin plasma concentrations. A non-compartmental pharmacokinetic analysis was performed. The median Cmax was 4.0 μg/mL and 2.7 μg/mL for intramuscular and intranasal administration, respectively, while the median AUCinf was 6.9 h μg/mL for intramuscular administration and 4.9 h μg/mL for intranasal administration. For both routes, the median Tmax was 0.2 h, and flunixin was detectable in some samples up to 60 h post-administration. Intranasal delivery had a relative bioavailability of 88.5%. These results suggest that intranasal flunixin has similar, although variable, pharmacokinetic parameters to the intramuscular route, making it a viable route of administration for use in grower swine.
As a pilot study, one sedated female Yorkshire cross grower pig (25.3 kg) was administered flunixin (Banamine®-S, Merck Animal Health, Madison, NJ, USA) at 3.0 mg/kg intranasally using an intranasal mucosal atomization device (MAD) (Teleflex, Morrisville, NC, USA) with 1 mL luer lock syringe to determine how drainage of the nasal sinus would produce higher concentrations of flunixin in the plasma collected from the jugular vein compared to the peripheral vein. Plasma samples were collected from femoral arterial catheter, jugular venous catheter and auricular arterial catheter at 0 (pre-treatment), 0.08, 0.25, 0.5, 0.85, 1, 1.5, 2, 2.5 and 3 h post-treatment. This study was approved by the North Carolina State University Institutional Animal Care and Use Committee (IACUC # 20-195-A). Figure 1 shows the plasma concentration of flunixin from each catheter site following the administration of 3.0 mg/kg IN flunixin from a pilot study. On NCA, the time to peak concentration (Tmax) was 0.25 h for all catheter sites. The peak concentration (Cmax) was 6.82 μg/mL for the jugular vein, 6 μg/mL for the auricular artery, and 5.17 μg/mL for the femoral artery. The area under the curve from time zero to the last time point (AUClast) for the jugular vein, the auricular artery and the femoral artery was 10.2, 9.7 and 9.3 h μg/mL, respectively. Thus, for ease of catheter maintenance and minimally invasive sampling, the jugular catheter site was elected for the full pharmacokinetic study (IN and IM pharmacokinetic cross-over study).
The IN and IM pharmacokinetic cross-over study was approved by the North Carolina State University Institutional Animal Care and Use Committee (IACUC #20–195). Six healthy female Yorkshire cross grower pigs (8 weeks of age, 18–22 kg) were individually housed in climate-controlled rooms and received 2.2 mg/kg flunixin (Banamine® -S, Merck Animal Health, Madison, NJ, USA) by either the IM or IN route. Following a 5-day washout period, the pigs were administered 2.2 mg/kg flunixin via the opposite route as a cross-over study. Intranasal administration was completed using a MAD600 MADgic laryngo-tracheal mucosal atomization device (Teleflex Medical, Morrisville, NC, USA). The luer lock syringe was attached to this device and the drug was placed into the nostril. Before administration, 0.22 mL of the drug was added to the device to account for dead space volume. After the IN administration, pigs were encouraged to keep their heads raised for approximately 10 seconds to prevent leakage of the drug out of the nasal cavity. The researcher (LS) got behind the pig and lifted its head with her hands. Intramuscular administration was performed in the omotransversarius muscle using a 20G needle. The pigs were monitored at least twice daily for general appearance, attitude, appetite and well-being throughout the study. Blood samples were collected at 0 (pre-treatment), 0.16, 0.33, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12, 18, 24, 36, 48 and 60 h post-treatment. Samples were processed within 1 h and plasma was stored at −80°C until analysis. Drug concentrations in plasma were determined using UPLC-MS/MS. The UPLC-MS/MS system consisted of an Acquity UPLC I class Binary Solvent Manager, Acquity UPLC sample Manager FTN and a Xevo TQD tandem mass spectrometer (Waters Corporation, Milford, MA, USA). Flunixin was extracted from plasma via solid phase extraction using oasis prime HLB 96 well μElution plate (Waters Corporation, Milford, MA, USA). The range of standard curve was 0.0001–0.1 μg/mL and 0.05–5 μg/mL (R ≥ 0.99) and the limit of quantification was 0.001 μg/mL and 0.1 μg/mL, respectively. The analytical assay was validated with 1.2%–5.1% of precision with 96.0%–110.0% of recovery (Appendix S1 and Table S1). A NCA of flunixin in plasma was performed using commercially available software (Phoenix WinNonlin™, version 8.3, Certara, St. Louis, MO, USA) to determine pharmacokinetic parameters for both routes. Only values above the limit of quantification were included in the final results and data analysis. The relative bioavailability was calculated by comparing plasma levels (AUCinf) given via IN route of administration with plasma levels (AUCinf) achieved by IM injection for each pig. The equation to calculate the relative bioavailability is described below: Relative bioavailability IN (%) = (AUCinf IN/AUC inf IM) × (Dose IM/Dose IN) × 100. Parameters (AUCinf, half-life, Cmax, Tmax and MRT) for each route of administration were compared with a Wilcoxon Matched Pairs Signed Rank Test using JMP® Pro Software version 16.0 (SAS, Cary, NC, USA). Differences were considered significant at p < .05. The statistical comparison results are shown in Table S2.
No major adverse effects on the pigs were observed following IN or IM administration of flunixin. Figure 2 shows flunixin concentration (mean ± standard deviation, μg/mL) versus time curve (h) following a single IM and IN administration of 2.2 mg/kg flunixin. The results of pharmacokinetic parameters for both IM and IN administration are shown in Table 1. Our study found a good median relative bioavailability (88.5%) with 39.1–113.8% range and identical Tmax (0.2 h) for the IN group compared to the IM group. The rich blood supply to the nasal mucosa likely allows for rapid absorption of flunixin with minimal swallowing or risk of first-pass hepatic metabolism (Enomoto et al., 2022; Hampton et al., 2021). In order to minimize loss of the drug from the nose, a commercially available laryngo-tracheal mucosal atomization device (MAD600 MADgic, Teleflex Medical, Morrisville, NC, USA) was used to administer the flunixin intranasally. The MAD atomizer is designed to create a fine mist of particles and ease the absorption of the drug through mucosa. The intranasal group had a median Cmax of 2.7 μg/mL that was significantly lower than that of IM group (4.0 μg/mL) (p = .047), however, both routes had similar ranges in plasma concentrations (0.001–10 μg/mL). Intranasal flunixin had a median half-life of 7.4 h, which was almost identical to that of IM flunixin (7.3 h). There was considerable variability observed in the plasma concentrations (Figure S1), pharmacokinetic parameters and relative bioavailability of IN flunixin between study pigs.
| Parameters | Intramuscular administration | Intranasal administration |
|---|---|---|
| λz (1/h) | 0.1 (0.07–0.13) | 0.09 (0.07–0.13) |
| HLλz (h) | 7.3 (5.3–9.6) | 7.4 (5.3–9.5) |
| Tmax (h) | 0.2 (0.2–0.3) | 0.2 (0.2–0.3) |
| Cmax (μg/mL) | 4.0 (2.9–5.8) | 2.7 (2.1–5.6) |
| AUClast (h μg/mL) | 6.9 (5.0–8.5) | 4.8 (3.0–8.9) |
| AUCinf (h μg/mL) | 6.9 (5.0–8.5) | 4.9 (3.0–8.9) |
| AUCextrap (%) | 0.3 (0.2–0.5) | 0.5 (0.1–0.8) |
| Relative bioavailability (%) | 88.5 (39.1–113.8) |
- Note: AUCextrap, extrapolation of AUC; AUCinf, area under the curve from time zero to infinity; AUClast, area under the curve from time zero to the last time point; Cmax, maximum concentration; HLλz, terminal half-life; Tmax, time to the maximum concentration; λz, elimination rate constant.
Many factors, including nasal tissue pH, presence of mucus, nasal anatomy and blood flow, drug administration technique, animal individuality and drug loss posteriorly into the oropharynx can influence drug absorption through the nasal mucosa (Enomoto et al., 2022). However, there were some limitations including a small sample size (n = 1 for the pilot study and n = 6 for the cross-over pharmacokinetic study) lacking the statistical power, sampling site (jugular vein) and healthy animal versus disease animal. The statistical power analysis (power: 80% and alpha: 0.05) was conducted using a sample size calculator (Clincalc.com). The results from our study were used for power analysis because the published pharmacokinetic data of IN flunixin was not available in grower pigs. This analysis revealed that a minimum of 26 grower pigs would be needed to have power for this study design. Additionally, the jugular vein was selected for sampling in a pharmacokinetic cross-over study to ease the maintenance of catheter throughout the study period based on pilot study data, thus a possibility of overestimation of drug concentration in the IN group was considered. However overall, IN route might be a vital addition to the swine industry by significantly improving animal welfare through providing fast and effective pain management during painful procedures. The intranasal route would be also expected to improve carcass quality by avoiding muscle damage caused by an injection and improve worker safety by minimizing the use of needles.
Further research is required to determine the analgesic efficacy of flunixin, and tissue residue study to estimate the drug withdrawal interval and pharmacokinetic study on farm. The neonatal piglets (10 days of age) are also considered as a target age group in order to obtain full pharmacokinetic profiles as well as to observe for any adverse effects to see if IN route can be the acceptable route of flunixin administration because legislation in Europe and Canada requires piglets to receive analgesia during processing due to welfare concerns, but no such legislation is in place in the United States.
Intranasal flunixin resulted in a similar range in plasma concentrations, but was highly variable, compared to IM flunixin. However quick absorption of flunixin was observed following IN administration, therefore this route might be another analgesic option as minimally invasive and needless drug delivery route in healthy grower pigs.
AUTHOR CONTRIBUTIONS
EW contributed to the study design, sample collection and drafting of the manuscript. LS contributed to the study design, sample collection, writing and editing the manuscript. HE contributed to sample collection, assay development, sample analysis, pharmacokinetic analysis and writing and editing the manuscript. KM contributed to the study design, pharmacokinetic analysis, statistical analysis and editing the manuscript. RB contributed to editing the manuscript. All authors have reviewed and approved the final version of the manuscript.
ACKNOWLEDGEMENTS
This research was supported by the USDA National Institute of Food and Agriculture (grants no. 2019-41480-30292 and 2020-41480-23520; Kansas City, MO) which fund the Food Animal Residue Avoidance Databank (FARAD).
CONFLICT OF INTEREST STATEMENT
The authors have not stated any conflicts of interest.
ETHICS STATEMENT
The authors confirm that the ethical policies of the journal, as noted on the journal’s author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received. The authors confirm that they have adhered to US standards for the protection of animals used for scientific purposes.
